1. Field of the Invention
The present invention relates to an apparatus for detecting an atomic force in water, particularly an atomic force based on an electrostatic interaction.
2. Description of the Related Art
The development of a scanning tunneling microscopy (STM) has recently brought about a marked improvement in means for observing the microscopic surface structures of various types of sample surfaces. The STM is an instrument for imaging individual atoms on a sample surface one by one by monitoring tunnel currents flowing between the metal tip and the sample surface. The STM is basically used to observe metals and semiconductors having high conductivity. For samples having low conductivity, an atomic force microscopy (AFM) for detecting an atomic force acting between the tip and the sample surface is used to observe surface structures at the atomic level. Actually, the AFM is used to obtain an image of each atom on the surface of an ionic crystal such as NaCl or a lamellar substance such as mica or graphite.
Objects to be observed by the STM and the AFM are not limited to inorganic materials such as semiconductors and metals but tend to include organic molecules such as benzene and phthalocyanine and biological molecules such as proteins and DNAs. Attempts to observe the surfaces of such substances have been reported. Since there is no appropriate means for estimating the structures of biological molecules, the three-dimensional structures of many kinds of molecules are not yet known. If, therefore, surface structures can be detected by the STM and the AFM, it will greatly contributes to analysis of three-dimensional structures. In the state-of-the art techniques, however, observation of organic molecules and biological molecules by means of the STM and the AFM still entails various problems.
Organic molecular images obtained by the STM and the AFM are very obscure as compared with semiconductor and metal images which allow discrimination of the structure of each atom. Especially when a biological molecule is imaged, the overall structure image of the order of several nanometers can be only vaguely seen, but the structures of atoms cannot be discriminated from each other. In addition, reliable observation conditions are not established yet. For example, when a molecule surface is wet, an image can be obtained. If, however, the surface is dry, no image can be obtained. Furthermore, the reproducibility of images is poor.
The followings are considered as the reasons for such problems. It is basically difficult to apply the STM to samples other than those having high conductivity. Since organic molecules and biological molecules have low conductivity, theoretically, no tunnel currents should flow and no molecule images should be obtained. It therefore seems that obscure molecular structure images obtained by the STM up to the present are not attributable to tunnel currents between the tip and a molecule but are based on currents generated by some other factors. Although a definitive explanation for the generation mechanism of these currents is not yet forthcoming at present, it is highly probable that the generation of the currents is associated with thin water layers and the like which are considered to exist on molecular surfaces. Even if currents generated by such a mechanism are monitored, it is impossible to detect molecular structures at the atomic level.
Currently, the AFM is operated under the condition that a large repulsive force of 10.sup.-7 to 10.sup.-8 N acts between the tip of the cantilever and a sample. However, since an organic molecule or a biological molecule is structurally weaker than an inorganic material, if the AFM is operated under the above-mentioned condition, there is a high possibility that structural deformation or destruction of the molecule is caused. Therefore, even if an image is obtained, it is impossible to distinguish whether the image represents the inherent structure of the molecule or the structure deformed by the repulsive force.
As described above, although attempts are made to observe the structures of organic molecules and biological molecules by using the STM and the AFM, these instruments are not basically suitable for the estimation of those molecules at the atomic level. Therefore, demands have arisen for means capable of estimating molecular structures at the atomic level on the basis of the principles applicable to the physical properties of molecules.
A very distinct characteristic feature associated with the structures and physical properties of organic molecules and biological molecules is that these molecules contain functional groups, and the physical properties and functions of the molecules are greatly influenced by the bonding positions of the functional groups. Typical functional groups are --COOH, --NH.sub.2, --OH, --CONH.sub.2, --SH, and the like. A functional group is a minimum unit exhibiting the function of a molecule. The function of a functional group is determined by its electrical property, more specifically a charged or polarized state. For example, the group --COOH is an acidic functional group which tends to be negatively charged to become --COO.sup.- in water. The group --NH.sub.2 is a basic functional group which tends to be positively charged to become --NH.sub.3.sup.+ in water. Although the groups --OH, --CONH.sub.2, and --SH are not apt to be ionized in water, these functional groups themselves have large dipoles.
Since the functional groups are ionized or have large dipoles in this manner, they cause a strong electrostatic interaction with their surroundings. A typical example of such an interaction is one caused by protein. Protein consists of 20 types of amino acids. The amino acids are combined to each other to constitute a one-dimensional chain structure, and this chain structure constitutes a complicated stereoscopic structure. In water in which a protein exhibits its function, the functional groups of the amino acids being ionized or having large dipoles are exposed on the protein surface. It is considered that the charge distribution based on the functional groups is closely related to the stereoscopic structure of the protein or the molecular function inherent in the protein, typically an enzyme reaction. In spite of the fact that the charge distribution on a protein surface and the surrounding electric field distribution based on the charge distribution are important, there are no measurement means for such distributions.
A great deal of attention is paid to techniques associated with the STM and the AFM as promising candidates for means for measuring an intramolecular charge distribution based on functional groups in a solution and a surrounding electric field distribution at the atomic level. For example, electrochemical AFM (S. Manne et al., Science, 251, 183-186 (1991)), EMF (electrostatic force microscope) (B. D. Tetris et al., Phys. Rev. Lett., 63, 2669-2672 (1989); B. D. Tetris et al., J. Vac. Sci. Tech., A8(1), 374-377 (1990)), and IFM (interfacial force microscope) (S. A. Joyce et al., Rev. Sci. Instrum., 62, 710 (1991)) have been proposed. These associated techniques, however, cannot achieve the above object for the following reasons.
The electrochemical AFM is an instrument designed such that an electrode system electrochemically controlled is formed in water to observe the microscopic structure of a metal sample surface. In the electrochemical AFM, a three-electrode system is constituted in a sample cell by a metal sample as a working electrode, a stainless clip as a counter electrode which holds a cantilever, and a copper wire as a reference electrode, and the potential of the metal sample surface is controlled by the three-electrode system. Similar to the general AFM, this electrochemical AFM is designed to detect a repulsive force acting between the cantilever and a metal sample surface but cannot measure a charge distribution or a potential distribution on the sample surface. In addition, since the copper wire is used as the reference electrode, currents flow between the reference electrode and the working electrode and between the reference electrode and the counter electrode. This makes it difficult to perform accurate potential control, and various kinds of electrode reactions are caused.
The EFM is designed to measure an electrostatic force acting between a metal cantilever and a metal sample surface. The EFM, however, is designed to perform measurement in the air or in a vacuum, and does not have a cell for holding the cantilever and the sample in water. In addition, in the EFM, the cantilever is vibrated at a frequency near its resonance frequency, and the electrostatic force is detected on the basis of changes in amplitude, phase, and frequency of the vibrations. The reason why the method of vibrating the cantilever is employed is that the EFM is designed to detect a force acting in a region located 10 to 100 nm apart from the sample. In water, however, since water has a high relative dielectric constant of 80, the electrostatic force is very small in a region located 10 nm or more apart from the sample. For this reason, in water, the force must be detected in a region apart from the sample by only several nanometers. In this case, the cantilever vibration method cannot be used because the cantilever may collide with the sample. Furthermore, in the EFM, a voltage is applied between the metal cantilever and the metal sample. If this method is used in water, since water has high conductivity, a current flows in water. Consequently, various kinds of electrode reactions, e.g., the decomposition of water on the sample surface and the cantilever surface, are caused to hinder detection of the electrostatic force. Therefore, when the electrostatic force is to be detected in water, a method in which no current flows upon application of a voltage must be employed.
The IFM is an apparatus designed to measure a minute force, especially an attractive force, acting between the tip and the sample which are spaced apart by a short distance of a few nanometers or less. In an AFM of ordinary type (including an EFM and an electrochemical AFM), when an attractive force acts between the tip and the sample, the cantilever will be pulled toward the sample, causing a phenomenon known as "jump-in," and will come into contact with the sample, if the gradient of the attractive force surpasses the spring constant of the cantilever. The range over which the attractive force can be measured is inevitably limited. With the IFM it is possible to measure an attractive force built up between the tip and the sample which are spaced apart by such a short distance, without causing jump-in. This is because a capacitor is provided, one of the electrodes of which is the back surface of the cantilever, and the attractive force between these electrodes is controlled by voltage, thereby canceling out the tip-sample attractive force. An electrostatic force between the tip and the sample has not been measured in the IFM-related experiment and research hitherto reported. It is, nonetheless, possible to measure an electrostatic force in the atmosphere or in a vacuum if a metal tip is used as in the EFM. If the IFM is used in an aqueous solution, however, the attractive force acting between the electrodes of the capacitor, which are spaced apart by a few tens of microns, is screened by water and thereby decreases to so small a value that it can no longer cancel out the tip-sample attractive force. The IFM has another problem that a current flows between the electrodes of the capacitor, resulting in various electrode reactions on the electrode surfaces, which in turn render the IFM operation unstable. Hence, it is basically problematical to employ the IFM in a solution in order to evaluate intramolecular charge distribution based on functional groups.
As described above, at present, there is no appropriate means for estimating the structures of organic molecules and biological molecules at the atomic level. In spite of the fact that the functional group distribution and charge and potential distributions based on the functional groups play important roles in terms of the physical properties and functions of the molecules especially in a solution in which the molecules exhibit their functions, there is no means for measuring them. Although techniques associated with the STM and the AFM are promising as means for estimating the functional group distribution, the charge distribution, and the potential distribution, they have various kinds of problems and hence cannot achieve the above-described object.